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Combination of linkage and association mapping with genomic prediction to

infer QTL regions associated with gray leaf spot and northern corn leaf blight
resistance in tropical maiz...

Article in Frontiers in Genetics · November 2023

DOI: 10.3389/fgene.2023.1282673

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 TYPE Original Research
PUBLISHED 07 November 2023
DOI 10.3389/fgene.2023.1282673

Combination of linkage and

OPEN ACCESS association mapping with
EDITED BY
Krishnanand P. Kulkarni,
Delaware State University, United States
genomic prediction to infer QTL
REVIEWED BY
Qin Yang,
regions associated with gray leaf
Northwest A&F University, China
Zhenhua Wang,
Northeast Agricultural University, China
spot and northern corn leaf blight
*CORRESPONDENCE
Mathews M. Dida,
resistance in tropical maize
mitodida@yahoo.com
Manje Gowda,
m.gowda@cgiar.org
Dennis O. Omondi 1,2, Mathews M. Dida 1*, Dave K. Berger 3,
RECEIVED 24 August 2023
Yoseph Beyene 4, David L. Nsibo 3, Collins Juma 4,2,
ACCEPTED 18 October 2023 Suresh L. Mahabaleswara 4 and Manje Gowda 4*
PUBLISHED 07 November 2023
1
Department of Crops and Soil Sciences, School of Agriculture, Food Security and Environmental
CITATION
Sciences, Maseno University, Kisumu, Kenya, 2Crop Science Division Bayer East Africa Limited, Nairobi,
Omondi DO, Dida MM, Berger DK,
Kenya, 3Department of Plant and Soil Sciences, Forestry and Agricultural Biotechnology Institute (FABI),
Beyene Y, Nsibo DL, Juma C,
University of Pretoria, Pretoria, South Africa, 4The Global Maize Program, International Maize and Wheat
Mahabaleswara SL and Gowda M (2023),
Improvement Center (CIMMYT), Nairobi, Kenya
Combination of linkage and association
mapping with genomic prediction to infer
QTL regions associated with gray leaf
spot and northern corn leaf blight
resistance in tropical maize.
Front. Genet. 14:1282673. Among the diseases threatening maize production in Africa are gray leaf spot (GLS)
doi: 10.3389/fgene.2023.1282673 caused by Cercospora zeina and northern corn leaf blight (NCLB) caused by
COPYRIGHT
Exserohilum turcicum. The two pathogens, which have high genetic diversity,
© 2023 Omondi, Dida, Berger, Beyene, reduce the photosynthesizing ability of susceptible genotypes and, hence, reduce
Nsibo, Juma, Mahabaleswara and Gowda. the grain yield. To identify population-based quantitative trait loci (QTLs) for GLS
This is an open-access article distributed
under the terms of the Creative and NCLB resistance, a biparental population of 230 lines derived from the tropical
Commons Attribution License (CC BY). maize parents CML511 and CML546 and an association mapping panel of
The use, distribution or reproduction in 239 tropical and sub-tropical inbred lines were phenotyped across multi-
other forums is permitted, provided the
original author(s) and the copyright environments in western Kenya. Based on 1,264 high-quality polymorphic
owner(s) are credited and that the original single-nucleotide polymorphisms (SNPs) in the biparental population, we
publication in this journal is cited, in identified 10 and 18 QTLs, which explained 64.2% and 64.9% of the total
accordance with accepted academic
practice. No use, distribution or phenotypic variance for GLS and NCLB resistance, respectively. A major QTL
reproduction is permitted which does not for GLS, qGLS1_186 accounted for 15.2% of the phenotypic variance, while
comply with these terms. qNCLB3_50 explained the most phenotypic variance at 8.8% for NCLB
resistance. Association mapping with 230,743 markers revealed 11 and 16 SNPs
significantly associated with GLS and NCLB resistance, respectively. Several of the
SNPs detected in the association panel were co-localized with QTLs identified in
the biparental population, suggesting some consistent genomic regions across
genetic backgrounds. These would be more relevant to use in field breeding to
improve resistance to both diseases. Genomic prediction models trained on the
biparental population data yielded average prediction accuracies of 0.66–0.75 for
the disease traits when validated in the same population. Applying these prediction
models to the association panel produced accuracies of 0.49 and 0.75 for GLS and
NCLB, respectively. This research conducted in maize fields relevant to farmers in
western Kenya has combined linkage and association mapping to identify new
QTLs and confirm previous QTLs for GLS and NCLB resistance. Overall, our

Frontiers in Genetics 01 frontiersin.org

Omondi et al.
findings imply that genetic gain can be improved in maize breeding for resistance to
multiple diseases including GLS and NCLB by using genomic selection.
KEYWORDS
maize, gray leaf spot, northern corn leaf blight, quantitative trait loci, association mapping,
genome-wide association study
1 Introduction
Despite its importance, maize production in Kenya is still low
with an estimated average production of 1.8 t/ha-1 among
smallholder farmers when compared to the country’s potential
production yield of 6 t/ha-1 (Munialo et al., 2019). This is partly
due to the threat of highly destructive and virulent fungal pathogens
limiting crop production (Beyene et al., 2019). In this context,
northern corn leaf blight (NCLB), also known as northern leaf
blight (NLB) or Turcicum leaf blight (TLB), caused by Exserohilum
turcicum (Pass.) (Leonard and Suggs, 1974), and gray leaf spot (GLS)
caused by Cercospora zeina Crous & U Braun (Crous et al., 2006) on
the African continent (Nsibo et al., 2019; Nsibo et al., 2021) are the
most lethal and economically significant foliar diseases of maize
(Beyene et al., 2019; Sserumaga et al., 2020).
The two diseases reduce the photosynthetic potential of a plant
and eventually decrease the grain yield (Saito et al., 2018). GLS is
characterized by tan-to-gray rectangular lesions that are limited
within the leaf veins (Korsman et al., 2012). It is associated with yield
losses of approximately 30%–50%, particularly when using
susceptible lines (Kinyua et al., 2010). On the other hand, NCLB
is characterized by long elliptical cigar-shaped lesions on leaves that
are gray–green (Welz, 1998). NCLB or TLB has been reported to
cause yield reductions of 36%–72% in susceptible maize genotypes
(Berger et al., 2020). The high genetic diversity reported for C. zeina
and E. turcicum in Kenya (Borchardt et al., 1998; Nsibo et al., 2021)
could lead to recombining pathogen populations, hence posing a
greater risk to the vulnerable susceptible lines (McDonald and
Linde, 2002). Therefore, there is a need to continuously discover
new sources of resistance.
The doubled haploid (DH) technology offers the fastest
alternative to achieve 100% homozygosity (attained in two
generations) which is essential for a mapping population and
population improvement (Prasanna et al., 2021). To complement
the DH technology, genotyping by sequencing platforms such as
Diversity Arrays Technology (DArT) offers a high-throughput
platform for genotyping single-nucleotide polymorphism (SNP)
markers (Kilian et al., 2012; Sansaloni et al., 2020). The DArTseq
platform is purposefully a powerful tool for genome-wide discovery
of SNP markers without prior sequence information (Wenzl et al.,
2004). In addition, it generates high-density linkage maps, and it is
also cost-competitive (Jaccoud et al., 2001; Sánchez-Sevilla et al.,
2015).
Complex traits such as GLS and NCLB resistance are
controlled by polygenic genes with minor effects that are
distributed throughout the genome (Welz and Geiger, 2000;
Wisser et al., 2006; Poland et al., 2011; Van Inghelandt et al.,
2012; Chen et al., 2015; Ding et al., 2015). Mapping of the
quantitative trait loci (QTLs) based on linkage analysis is a
powerful tool for identifying the genomic regions associated
Frontiers in Genetics
10.3389/fgene.2023.1282673
with the traits of interest. Previous QTL studies have mapped
QTL for resistance to GLS and NCLB on all 10 maize
chromosomes (Berger et al., 2014; Chen et al., 2015). A
number of these QTLs have been fine-mapped with others
cloned and the molecular mechanisms underlying such QTL
characterized. Additionally, QTL mapping offers the advantage
of mapping as early as at the F2 populations; however, this is
characterized by the limited number of recombination events
captured and sizeable confidence interval (Challa and Neelapu,
2018; Rashid et al., 2020).
Genome-wide association studies (GWAS) attempt to overcome
the drawbacks of QTL mapping as they utilize age-old
recombination events in a large array of unrelated individuals
leading to high-speed decay of linkage disequilibrium (Xiao et al.,
2017; Kolkman et al., 2020). GWAS studies dig into the entire
genome of different varieties (considering the SNPs present in the
genotypic data) to establish the link between genotypic variations
and the corresponding trait (Challa and Neelapu, 2018). Kibe et al.
(2020a) combined the use of linkage mapping and GWAS to detect
the significant SNPs and QTL conditioning resistance to GLS in an
Improved Maize for African Soils (IMAS) diversity panel and a set of
DH populations in Kenya. Several putative candidate genes involved
in the transportation channel were identified to have a role in plant
defense. In the present study, we attempted to validate some of the
GLS resistance QTLs reported in the study by Kibe et al. (2020a) by
using a common tropical parent CML511.
Genomic prediction (GP) is another promising genomic tool
that has been applied successfully in plant breeding programs
(Crossa et al., 2017). Previous studies indicated the potential of
GP to increase genetic gain and reduce the time taken in breeding
programs significantly (Beyene et al., 2019; Kibe et al., 2020a;
Kibe et al., 2020b). In contrast to genetic mapping which
identifies significant marker–trait associations, GP uses all
markers available to estimate their effects, thus providing a
powerful approach to account for any effects that might have
been missed by either genetic or association mapping (Beyene
et al., 2019). GP exhibits a close relationship to GWAS owing to
the large genomic and phenotypic datasets used by the methods
(Beyene et al., 2021).
However, this does not mean the complete withdrawal of genetic
mapping but rather the incorporation of the two in genetic studies as
complementary approaches since each provides considerable
advantages. With this background, the objectives of this study
were as follows: (1) to phenotypically characterize an elite
tropical DH population and 240 tropical and sub-tropical maize
inbred lines panel for their responses to GLS and NCLB, including
correlation with other agronomic traits; (2) to identify population-
based common QTL regions and significant SNPs using GWAS and
linkage mapping; and (3) to assess the usefulness of GP in breeding
for GLS and NCLB resistance in tropical maize.
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2 Materials and methods
2.1 Study sites and genetic material
This study used (i) a biparental DH population derived from the
tropical×tropical germplasm CML511×CML546 inbred lines and
(ii) an association panel made up of a collection of 239 tropical and
sub-tropical maize inbred lines with early and intermediate maturity
in Eastern Africa, representing some of the genetic diversity (for low
N, drought, and biotic stresses, Beyene et al., 2021; Prasanna et al.,
2021). The association panel was evaluated in three locations in
western Kenya, at Kitale (1.0191° N and 35.0023° E, 1900 masl);
Shikutsa (0°16′57.83″N and 34°45′6.71″E, 1561 masl); and
Kakamega (0°17′3.19″N and 34°45′8.24″E, 1535 masl). The
biparental population was evaluated across different ecologies in
western Kenya; Maseno University field demonstration site in 2018
(0°00′18.2″S and 34°35′43.5″E, 1500masl), Maseno 2019
(0°00′07.0″S and 34°35′41.9″E, 1503 masl), and farmer’s field in
Kabianga 2018 (0°25′24.1″S and 35°07′31.7″E, 1780 masl).
2.2 Experimental design
Two hundred and thirty (230) entries (228 DH lines and two
parents) of the biparental population were planted in a 5 × 46 alpha
lattice design, randomized, and replicated three times at each site by
using the CIMMYT’s field book (Vivek et al., 2007). The association
panel was also planted in 5 × 48 alpha lattice design, randomized,
and replicated two times in each of three environments.
Experimental plots consisted of 3 m long single rows with the
rows spaced at 0.6 m apart. Adjacent plots were planted 0.75 m
apart with an alley of 1.2 m at the end of each plot. Each plot was
planted with 13 hills, with two seeds getting planted per hill.
Thinning was later conducted to one plant per hill. Border rows
of susceptible genotypes were also planted to act as spreaders of the
pathogen. The experimental plots were managed using standard
agronomic practices.
2.3 Phenotypic evaluation and data
collection
GLS and NCLB disease severity (DS) were scored on a per-
row basis using an ordinal 1–9 scale adapted from the work of
Berger et al. (2014) for GLS and Berger et al. (2020) for NCLB. For
DH population, DS ratings for GLS and NCLB were taken once
per week for at least 5 weeks starting on average at 15 days after
flowering (R2; reproductive stage two). All the data were
collected using the CIMMYT’s field book (Vivek et al., 2007).
The DS scale was used as follows: score 1 for no GLS or NCLB
lesions visible on the entire plant, 2 indicated close inspection of
each leaf is necessary to find lesions, 3 indicated lesions are more
easily seen but are majorly restricted to leaves lying below the
ears, 4 indicated individual lesions are just becoming visible on
the ear leaf and the leaves above the ears, 5 indicated lesions are
more visible on the leaves above the ears, with the infections
capturing <10% of the top leaves, 6 indicated lesions are more
easily seen on the leaves above the ear leaf with infections
Frontiers in Genetics
10.3389/fgene.2023.1282673
covering >10% of the leaf area, 7 for GLS and NCLB lesions
dominating the leaf area on all the leaves with 50% of the maize
leaf surface diseased, 8 for GLS and NCLB lesions prevalent on all
the leaves of the maize plant with 80% of the maize leaf surface
diseased, and 9 for GLS and NCLB lesions prevalent on all the
leaves of the maize plant with the maize plant exhibiting a gray
appearance with >80% of the maize leaf area diseased. For both
GLS and NCLB, the DS scores over five intervals were used to
calculate the area under the disease progress curve (AUDPC,
Shaner, 1977). For the GWAS panel, both DS data were recorded
at the dough stage of the plants. For DH population, data were
also collected on days to anthesis (AD, the number of days from
planting to when 50% of the plants in a plot were shedding
pollen), days to silking (SD, the number of days from planting to
when 50% of maize crops in a plot were showing silk), plant
height (PH, cm), and ear height (EH, cm). Maize development
stages were recorded using the scale of Purdue University (http://
extension.entm.purdue.edu/fieldcropsipm/corn-stages.php).
2.4 Statistical analysis of the phenotypic data
Multi-environment trial analysis with R for windows
(META-R) version 6.0 (Alvarado et al., 2015) was used to
obtain the best linear unbiased estimations (BLUEs) and best
linear unbiased predictions (BLUPs). In addition to BLUEs and
BLUPs, META-R was also used to compute the genetic
correlations among all the variables and among environments,
least significant difference (LSD), grand mean, variance
components, coefficients of variation (CV), and broad-sense
heritability for all the variables. Analysis of the phenotypic
data for both biparental population and association panel was
conducted both within and across environments.
The BLUEs and the BLUPs were calculated for DS of GLS and
NCLB, PH, EH, AD, SD, and the AUDPC which were the response
variables. The columns in the input files were selected to be the
factor names with the environment, replicate, block, and genotype as
the independent variables. For analysis across environments using a
lattice design, the following linear mixed model was used.
Yijkl = µ +Envi + Repj(Envi) +Blockk(Envi Repj) +Genl +
Envi ×Genl + εijkl.
From the aforementioned equation, Yijkl represents the
performance of the trait of interest, µ corresponds to the all-
inclusive mean, Envi represents the effect of the ith environment,
Repj(Envi) represents the effect of the jth replication within the
ith environment, Blockk(Envi Repj) represents the effect of the
kth incomplete block within the jth replication in the ith
environment, Genl represents the effect of the lth genotype,
Envi ×Genl represents the environment by genotype
interaction, and εijk is the error variance. When calculating the
BLUEs, genotypes and covariates were considered fixed effects of
the model while other terms were included as random effects of
the model. The covariate was considered as fixed effect of the
model while all other terms were included in the random effects
of the model to estimate the BLUPs. Heritability for the different
traits was calculated as the ratio of the estimated genotypic
variance to the estimated phenotypic variance (Knapp et al.,
1985).
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2.5 Genotyping and QTL mapping
The CML511×CML546 DH and parental lines were grown in a
greenhouse. Maize leaf tissue samples were collected from young,
healthy seedlings at the V3 stage (3–4 weeks old), stored at −80°C,
and later freeze-dried for 72 h. High-quality genomic DNA was
isolated from freeze-dried tissues using the standard CIMMYT
laboratory protocol (CIMMYT, 2005). The DH lines were
genotyped using DArTSeq in Canberra, Australia. Approximately
15,000 SNPs were used for further quality checks (Murithi et al.,
2021). Trait Analysis by aSSociation Evolution and Linkage
(TaSSEL) (Bradbury et al., 2007) was used to summarize
genotype data by site, determine the allele frequencies, and
implement quality screening. All SNP variants that were
monomorphic between the two parents, had heterozygosity
of >0.05 and a minor allele frequency of <0.05, were filtered, and
1,264 high-quality SNPs were retained for QTL mapping.
Redundant markers were removed using the BIN tool in QTL
IciMapping v.4.2 (Meng et al., 2015). In the parameter setting
window for general information, eight functionalities were used
to define the mapping population. In the indicator row, 1 was
selected to denote QTL mapping in actual populations and 3 as the
population type as this study used a DH population. Kosambi was
set as the mapping function, marker position as the marker space
type, 10 as the number of chromosomes, 230 as the size of the
mapping population, and 6 as the number of traits.
The number of markers in each chromosome was specified in
the chromosome information part. The scores for all the DArTseq
markers were transformed into genotype codes following the scores
of the parents (2 denoted the marker type of the first parent,
0 denoted the second parent, 1 for the F1 marker type,
and −1 for the missing markers) (Meng et al., 2015). The genetic
linkage maps were constructed using the MAP functionality of QTL
IciMapping v.4.2 (http://www.isbreeding.net). Three steps were
followed in linkage map construction: grouping, ordering, and
rippling. The logarithm of odds score was set at 3.0 for grouping.
Ordering was performed using the ordering instruction with the
nnTwo Opt algorithm. The sum of adjacent recombination
frequencies (SARF) as the criterion and window size of 5 as the
amplitude were used to ripple the marker sequence and to fine-tune
the chromosome orders. All the outputting functionalities were
checked, and the map was drawn using the MAP functionality
(Meng et al., 2015).
In the phenotypic data, the BLUPs for the different traits were
used as the input files for QTL identification across environments
(Littell et al., 2007). The input file was loaded onto the project of
IciMapping v.4.2 (Meng et al., 2015). In the parameter setting
window ICIM-ADD, other parameters, such as step in scanning
represented by cM and stepwise regression of phenotype on marker
variables, were defined. An LOD threshold of 3.0 and
1000 permutations at α = 0.01 were set to declare the significant
QTL. The percentage of total phenotypic variance explained by an
individual QTL was determined using stepwise regression. To
ascertain the actual locations of the QTL for all the traits on the
chromosomes, the physical position of the identified QTL was
assigned based on the known physical position of the linked
markers and also available at the maize genetics and genomics
database (http://www.maizegdb.org/data_center/map), as described
Frontiers in Genetics
10.3389/fgene.2023.1282673
by Berger et al. (2014). The individual QTLs were assigned names
according to the QTL, trait name, chromosome, and marker
position, as described in the work of Kibe et al. (2020a).
2.6 Genotyping and association mapping
The DNA of all 239 inbred lines of the association panel was
extracted from seedlings at the 3–4 leaf stage and genotyped using
the genotype-by-sequencing (GBS) platform at the Institute for
Genomic Diversity, Cornell University, Ithaca, United States,
using high-density markers, as per the procedure described by
Elshire et al. (2011). SNP calling and imputation were conducted
at Cornell University. For SNP calling, raw data in a FASTQ file
together with the barcode information and Tags On Physical Map
(TOPM) data, which had SNP position information, were used. We
used TOPM data from AllZeaGBSv2.7 downloaded from Panzea
(https://www.panzea.org/), which contained information for
955,690 SNPs mapped with B73 AGPv2 coordinates. SNP calling
was then performed using the TASSEL-GBS pipeline (Glaubitz et al.,
2014; Wang et al., 2020). Using TASSEL ver5.2 (Bradbury et al.,
2007), SNPs with a heterozygosity of >5%, MAF of >0.05, and
minimum count of 90% were excluded by filtering from raw GBS
datasets, and 230,743 high-quality SNPs were retained for further
analysis in the association panel. To explore the population structure
and the ultimate number of subpopulations, principal component
analysis (PCA) as described by Price et al. (2006) was conducted in
TASSEL using SNPs across all panels. The first three principal
components were instrumental to visualize the existing
population stratification within the association panel, and this
was clearly displayed in a 3D plot. The PCA plots of the
association panel were computed using 230,743 SNPs; we then
plotted the variance (y-axis) against the principal components
(x-axis) to estimate the number of clusters within the population
(Sanchez et al., 2018). The data point at which the increase in the
number of principal components did not result in an increase in
variance (leveling off) indicated the number of subgroups within the
panel. To estimate the amount of genetic relatedness among
individuals, a kinship matrix was explored.
GWAS was performed with different models to compare and
choose the appropriate model with relatively less false positives. To
detect marker–trait associations, GWAS was performed using the
following models: (1) mixed linear models (MLMs; PCA + K + G)
that incorporated PCA, kinship (K), and genotypic data as
covariates; (2) the general linear model (GLM; PCA + G) which
incorporated the genotype data (G) and the PCA (Q) that both acted
as fixed effects to correct for the population structure; and (3) Fixed
and random model Circulating Probability Unification (FarmCPU),
in which the kinship (random) and the three-component analysis
(fixed) were identified as covariates (Lipka et al., 2012). Single-locus
GWAS models such as the GLM are characterized by high false
positive rates, as a complementary model, and the MLM utilizes the
Bonferroni correction to overcome the challenge of false positive
rates and identify the loci of interest (Khan et al., 2021). The software
TASSEL (Bradbury et al., 2007) was instrumental to run the GLM +
PCA and MLM. The − log 10 p values for all the analyzed SNPs for
both GLS-DS and NCLB-DS were used to construct the Manhattan
plots. Q–Q plots were plotted from the estimated -log10 (p) from the
04 frontiersin.org
Omondi et al.
association panel for GLS-DS and NCLB_DS traits. Analysis of the
association panel was conducted in TASSEL based on
230,743 filtered SNPs. The R package ‘FarmCPU’ with the
Genome Association and Prediction Integrated Tool (GAPIT)
was used for GWAS analysis (Tang et al., 2016). The false
discovery rate (FDR) was calculated for significant associations
using the Benjamini and Hochberg (1995) correction method,
with 8 × 10−5 as the threshold. To summarize GWAS results per
chromosome, Manhattan scatter plots were generated by plotting
genomic positions of the SNPs against their negative log base 10 of
the p-values obtained from the GWAS model, with the F-test for the
null hypothesis on the y-axis.
SNPs detected in the association panel were examined as
polymorphisms in linkage disequilibrium with putative candidate
genes from the B73 reference gene set (https://phytozome-next.jgi.
doe.gov/jbrowse/index.html?data; Zea mays Zm-B73-REFERENCE-
NAM-5.0.55) (Goodstein et al., 2012). Putative candidate genes were
selected by delving into the information from Gene Ontology, Kyoto
Encyclopedia of Genes and Genomes (KEGG), and protein families
(Pfam) (Ashburner et al., 2000; Kanehisa and Goto, 2000; Bateman
et al., 2004).
Genomic prediction was carried out with ridge regression BLUP
(Zhao et al., 2012) within a biparental DH population for GLS,
NCLB, and agronomic traits at five-fold cross-validation. The
BLUEs across environments were used for the analysis. The same
set of 1,264 high-quality uniformly distributed SNPs with no
missing values and MAF>0.05 were used. For the maize
association panel, quality screening criteria of SNPs with
MAF >0.10 and no missing values were applied, and finally,
8,365 SNPs from the 230,743 SNPs were retained for the
analyses. The prediction was ‘within population’, where training
and validation sets were derived from within the biparental
population. For each trait, 100 iterations were carried out for the
sampling of the training and validation sets. The prediction accuracy
was calculated as the correlation between the observed phenotypes
and genomic estimated breeding values (GEBVs) divided by the
square root of heritability (Dekkers, 2007).
3 Results
3.1 Phenotypic data
As expected for western Kenya, there was high natural disease
pressure for both NCLB and GLS for all field trials of the biparental
CML511×CML546 DH population (Figure 1A), as well as the
association panel (Figure 1B). DS scores for both NCLB and GLS
were highest at the final disease rating time point in all field trials,
which corresponded in most cases to the last disease rating time
point (Supplementary Table S1). A significant difference in
resistance to NCLB was reported for the two parents
CML511 and CML546 (p-value = 0.011831, α < 0.05). Our data
show that CML511 is moderately susceptible and CML546 is more
resistant to NCLB. On the other hand, the two parents differed
slightly but not significantly in resistance to GLS (p-value =
0.200588, α < 0.05). For GLS, CML511 had an average score of
2.47 at the final rating, while CML546 had an average score of 4.0 at
the final average DS score (Supplementary Figure S1). These
Frontiers in Genetics
10.3389/fgene.2023.1282673
evaluations show that CML511 is resistant and CML546 is
moderately susceptible to GLS. A large portion of the biparental
population was extensively blighted by NCLB. Transgressive
segregation was observed in the population for GLS, NCLB, and
AD, as some of the genotypes were more resistant or susceptible
compared to the parental lines in the biparental population
(Figure 1).
The frequency distribution of GLS DS scores was fairly skewed
toward resistance in the biparental population (Figure 1A). The
frequency distribution of the DS scores for NCLB in the biparental
population followed an approximately normal distribution, as
shown in Figure 1A. The wide segregation of DS and AUDPC
scores for NCLB provided more evidence for the quantitative
control of resistance. The DH population also exhibited
continuous distribution for the days to anthesis, days to silking,
plant height, and ear height (Figure 1A). The GLS and NCLB DS
scores for the maize association panel ranged from 1.5 to 6
(Figure 1B), which were similar to the biparental DH population
scores, although the association panel trended toward higher GLS
and lower NCLB scores. On the other hand, the use of the nine-point
rating scale revealed extensive phenotypic variation in resistance to
GLS and NCLB across the association panel, with the panel
harboring more resistant lines (Figure 1B). The association panel
was also characterized by shorter days to anthesis compared to the
biparental population (Figure 1B).
3.2 Correlations between environments and
variables
In the DH population, the correlation between environments for
GLS DS was positive and highly significant (p < 0.001)
(Supplementary Table S2). A moderately high correlation was
observed between environments for NCLB DS scores
(Supplementary Table S2). The correlation across environments
for AD and SD was also highly significant at p < 0.001
(Supplementary Table S2).
The analyses of variance revealed significant genotypic and
genotype × environment interaction variances for GLS, NCLB
DS, and AUDPC values as well as other agronomic traits
(Table 1). Heritability estimates on an entry mean basis were
0.81, 0.81, 0.79, and 0.80 (Table 1) for GLS DS, AUDPC for
GLS, NCLB DS, and AUDPC for NCLB, respectively in the DH
population. However, the heritability estimates for DS in the
association mapping panel were lower (0.35 for GLS and 0.64 for
NCLB).
Interestingly, the correlation analyses in the DH population
showed low positive significant correlation between GLS (and
AUDPC_GLS) and NCLB (Figure 2), indicating that there are
different genomic loci that explain the variance for each disease.
The agronomic traits for reproductive traits, namely, flowering time
(AD) and days to silking (SD), were significantly negatively
correlated with DS and AUDPC for both GLS and NCLB
diseases (Figure 3). This indicated that maize lines with earlier
maturity had higher DS. As expected, ear height (EH) was highly
correlated with plant height (PH). GLS DS and AUDPC values were
weakly correlated with PH and EH, whereas NCLB DS and AUDPC
values were positively and significantly correlated with PH and EH
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Omondi et al. 10.3389/fgene.2023.1282673

FIGURE 1
Frequency distributions for GLS, NCLB disease, and other agronomic traits, namely, anthesis date, silking date, plant height, and ear height, evaluated
across the three locations in western Kenya. (A). Biparental CML511×CML546 DH population of 230 lines. (B). Association panel of 239 sub-tropical and
tropical maize lines across the three locations. DS scores were for the last rating time point.

TABLE 1 Estimates of means, components of genotypic (σ2G), genotype × environment interaction (σ2GxE), error variances (σ2e), and heritability (h2) for the
biparental CML511×CML546 DH population and the association panel evaluated across three environments each for GLS, NCLB, and other agronomic traits.

AD SD PH EH GLS GLS NCLB NCLB GLSa NCLBa

DS AUDPC DS AUDPC
Mean 81.74 82.69 127.89 55.02 2.36 52.27 4.58 108.46 3.11 3.62

σ 2
G 11.07** 14.37** 282.87** 67.79** 0.94** 382.44** 0.42** 311.71** 0.03* 0.07**

σ2GxE 2.37** 3.63** 16.66** 5.94** 0.49** 213.07** 0.19* 156.61** 0.09** 0.04**

σ2e 11.59 12.6 219.57 101.49 0.52 175.63 0.45 216.91 0.16 0.17

h2 0.84 0.85 0.90 0.84 0.81 0.81 0.79 0.80 0.35 0.64

LSD5% 2.69 3.04 11.21 7.07 0.84 17.31 0.6 15.95 0.33 0.52

CV 4.17 4.29 11.59 18.31 30.56 25.36 14.73 13.58 34.09 18.06

AD, days to anthesis; SD, days to silking; PH, plant height; EH, ear height; DS, disease severity on a scale of 1–9; GLS, gray leaf spot; AUDPC, area under the disease progress curve; NCLB;
northern corn leaf blight; CV, coefficient of variation; LSD, least significant difference; h2, broad-sense heritability; *, **significant at p=0.05 and 0.01 levels, respectively.
a
Disease severity scores of the maize association panel.

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Omondi et al. 10.3389/fgene.2023.1282673

TABLE 2 QTL detected by integrated composite interval mapping analysis for resistance to GLS and NCLB in the DH population evaluated in multiple locations.

Trait name QTL namea Chr Position (cM) Left markerb Right markerb LOD PVE (%) TPVE (%) Add Fav parent
GLS DS qGLS1_54 1 163 S1_283894617 S1_53456776 8.95 5.33 64.19 0.22 CML546

qGLS1_186 1 372 S1_190286762 S1_185978658 21.86 15.17 −0.28 CML511

qGLS1_185 1 383 S1_185978658 S1_143231392 11.34 9.01 0.21 CML546

qGLS2_30 2 208 S2_30710232 S2_32668550 3.79 2.24 0.11 CML546

qGLS3_151 3 92 S3_157562360 S3_150546157 5.31 3.37 0.13 CML546

qGLS5_07 5 189 S5_7548544 S5_1579511 3.62 2.25 −0.11 CML511

qGLS5_16 5 284 S5_15869219 S5_23093956 5.76 3.56 −0.14 CML511

qGLS7_158 7 105 S7_158889984 S7_158892468 4.49 2.58 −0.11 CML511

qGLS9_129 9 154 S9_135788881 S9_129671108 4.27 3.53 −0.13 CML511

qGLS10_50 10 217 S10_43765534 S10_54916081 3.19 6.21 0.22 CML546

NCLB DS qNCLB1_230 1 70 S1_229375633 S1_232878545 6.20 4.27 64.94 0.12 CML546

qNCLB1_303 1 429 S1_303106691 S1_304299395 3.13 2.08 0.08 CML546

qNCLB2_220 2 164 S2_223388206 S2_32056786 5.40 3.82 0.16 CML546

qNCLB3_02 3 163 S3_2734515 S3_1173815 4.95 3.57 0.11 CML546

qNCLB3_50 3 185 S3_65853211 S3_12761976 9.74 8.76 0.18 CML546

qNCLB4_200 4 272 S4_200040593 S4_201402668 3.50 4.58 −0.12 CML511

qNCLB5_83 5 80 S5_82971208 S5_160085856 3.60 4.56 0.13 CML546

qNCLB5_195 5 113 S5_194106967 S5_198705622 3.60 2.76 0.10 CML546

qNCLB6_137 6 204 S6_136207036 S6_137005821 3.21 2.17 −0.09 CML511

qNCLB6_153 6 335 S6_151834390 S6_153165363 3.95 3.58 −0.20 CML511

qNCLB7_120 7 96 S7_121214712 S7_47406965 7.51 5.58 −0.14 CML511

qNCLB7_174 7 257 S7_170932028 S7_174093748 3.43 2.44 −0.10 CML511

qNCLB8_171 8 276 S8_170418369 S8_171776990 7.85 5.77 0.14 CML546

GLS DS, gray leaf spot disease severity; NCLB DS; northern corn leaf blight disease severity; LOD, logarithm of odds; add, additive effect; PVE, phenotypic variance explained; fav parent,
parental genotype from where a favorable allele is contributing.
a
QTL, name composed by the trait code followed by the chromosome number in which the QTL was mapped and a physical location of the QTL. QTL names are italicized.
b
The exact physical position of the marker can be inferred from the marker’s name, for example, S1_82702920: chromosome 1; 82,702,920 bp (Ref Gen_v3 of B73).

(Figure 3). There were weak positive and significant correlations
between SD and PH/EH (Figure 3).
3.3 Construction of the genetic linkage map
and QTL analyses
The linkage map for the CML511×CML546 DH population was
constructed with a total of 1,264 SNP markers. The genetic linkage
map spanned a total map length of 3,344.9 cM with 2.65 cM as the
average distance between two adjacent markers. The linkage map, as
shown in Supplementary Figure S2, covered most of the maize
genome.
Several QTLs associated with resistance to GLS and NCLB with
small additive effects were detected through inclusive composite
interval mapping. QTL analyses revealed 10 QTLs distributed on
chromosomes 1, 2, 3, 5, 7, 9, and 10 for GLS DS which individually
explained 2.6%–15.2% of phenotypic variance and together
explained 64.19% of total phenotypic variance (Table 2). All
10 QTLs detected for GLS DS were also consistently detected for
GLS AUDPC values (Supplementary Table S3). For NCLB DS,
13 QTLs distributed in all chromosomes except for chromosomes
9 and 10, individually explained 2.2%–8.7% which together
contributed 64.94% of total phenotypic variation. AUDPC values
for NCLB revealed nine QTLs together explained 45% of total
phenotypic variance (Supplementary Table S3). Three QTLs on
chromosomes 3, 6, and 7 were consistent across NCLB DS and
AUDPC values. Among the agronomic traits, for AD, nine QTLs
detected together explained 63% of the total phenotypic variance,
and one QTL on chromosome 8 (qAD8_137) was a major effect QTL
which explained 15.84% of phenotypic variance ((Supplementary
Table S4). For SD, six QTLs were detected which together explained
60% of the total phenotypic variance. There were three QTLs on
chromosomes 1, 4, and 8 that were consistent for both AD and SD.
For PH, four QTLs were identified which together explained 52% of
total phenotypic variance. One major effect QTL (qPH8_129)

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Omondi et al.
FIGURE 2
Pairwise Pearson correlation analysis for eight traits evaluated in
three field trials in the biparental CML511×CML546 DH population. AD,
anthesis date; SD, silking date; GLS, gray leaf spot, NCLB, northern
corn leaf blight; AUDPC, area under disease progress curve; PH,
plant height; and EH, ear height. The x marks indicate values that are
not significant at p < 0.05.
detected on chromosome 8 explained 22% of phenotypic variance.
For EH, six minor effect QTLs and one major effect (qEH8_128)
QTL were detected which together explained 56% of total
phenotypic variance. QTL mapping predominantly revealed that
the additive gene effect defined the gene action for resistance to GLS
and NCLB.
3.4 GWAS analysis
After a quality check of GBS SNP markers, 230,743 SNPs were
retained for the final association analyses (Supplementary Figure
S3). The kinship matrix for these 239 lines was projected in the form
of a heatmap which shows the magnitude of the relationship
between the individuals (Supplementary Figure S4). This clearly
showed that there is no strong population structure in the
association panel used here. PCAs revealed five subpopulations
within the panel (Supplementary Figure S5).
Association analyses for GLS DS and NCLB DS data were
performed with GLM, MLM, and FarmCPU models (Figure 3).
For both GLS and NCLB DS traits, for the GLM model, the observed
p-values showed higher deviation from the expected p-values which
may cause high false positives. On the other hand, for the MLM
model, though the observed p-values were closer to the expected
p-values, overfitting of the model is observed. For the FarmCPU
model, the observed p-values were close to the expected p values and
were more effective in controlling the false positives (Figure 3). The
FarmCPU model is known to use both fixed and random effects
models iteratively which helps in avoiding overfitting of the model
by stepwise regression (Liu et al., 2016). Therefore, in this study, we
used the FarmCPU model in the association mapping.
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10.3389/fgene.2023.1282673
Association analyses revealed 11 and 18 SNPs significantly
associated with GLS DS and NCLB DS, respectively (Figure 3;
Table 3). For GLS-DS, the identified SNPs were distributed
across all chromosomes except for chromosomes 3, 6, and 7
(Figure 3). The Manhattan plot revealed that for GLS DS, the
highest peak was reported on chromosomes 4 and 8 (Figure 3A),
while for NCLB DS, the highest peak was reported on chromosome 8
(Figure 3C). Furthermore, we determined whether any of these
significant GLS or NCLB disease-associated SNPs identified in the
association analysis is co-located with the QTL for GLS or NCLB in
the biparental DH population. Two SNPs on chromosome 1 (S1_
192041854 and S1_253381765) were co-located with qGLS1_54
detected for both GLS DS and AUDPC values in the DH
population (Table 2; Table 3). Another SNP on chromosome 9
(S9_130213878) was found to be collocated within the qGLS9_129
and qG_AUDPC9_129 QTL region (Table 2; Table 3). Among the
16 SNPs identified for NCLB, marker S5_83980678 is found to be
within the region of NCLB DS QTL qNCLB5_83 (Table 2; Table 3).
To elucidate the molecular and physiological mechanisms
controlling GLS and NCLB DS, candidate genes were identified.
On all chromosomes, a total of 24 candidate genes were discovered
(Table 3). Four candidate genes closely associated with the SNPs for
GLS resistance were identified, namely, Zm00001eb077270,
Zm00001eb034870, Zm00001d017831, and Zm00001eb211960
(Table 3). There were six candidate genes with defense response
annotations that were associated with SNPs for NCLB resistance
(Zm00001eb201110, Zm00001eb035640, Zm00001eb232660,
Zm00001eb285080, Zm00001eb144960, and Zm00001d001787)
(Table 3).
Genomic prediction captures all variations from small to large
effects, which helps in improving complex traits. Prediction
correlations obtained from cross-validations are commonly used
to know the effectiveness of genomic predictions for different traits.
In this study, we applied genomic predictions within the DH
population and association panel for disease traits and also for
agronomic traits (Figure 4).
As expected, the mean prediction correlations for the DS traits
were higher in the DH population (GLS DS = 0.75, NCLB DS = 0.68)
than those in the association panel (GLS DS = 0.63, NCLB = 0.49)
(Figure 4). This was because of the highly relatedness among the DH
lines compared to the lines in the association panel. In addition,
relatively high-average prediction correlations were obtained for the
other traits when validated in the DH population, namely, 0.73, 0.66,
0.71, 0.72, 0.75, and 0.71 for GLS-AUDPC, NCLB-AUDPC, AD, SD,
PH, and EH, respectively.
4 Discussion
GLS and NCLB are economically important foliar diseases of
maize. Understanding their genetic basis of resistance is valuable to
designing an effective breeding strategy (Beyene and Prasanna,
2020). The DH population used in this study was artificially
inoculated with GLS; however, the locations evaluated were also
a hotspot for NCLB, so we observed both GLS and NCLB disease
symptoms in the same population. For early scoring, symptoms for
both diseases were clearly distinguishable, which helped to score the
data with more accuracy. Scoring at a later stage of disease
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FIGURE 3
(A, C) Manhattan plots for the GWAS of GLS and NCLB disease severity in the maize association mapping panel. The dashed horizontal line of the
Manhattan plot depicts the significance threshold value of p < 8 × 10−5. The x-axis indicates the SNP location along the 10 chromosomes, with
chromosomes separated by different colors. Q–Q plots (B, D) of the estimated -log10(p) from association panel for GLS-DS and NCLB_DS traits. The
black line bisecting the plot in Q–Q plots represents the expected p-values with no associations present. The blue line represents the observed
p-values using the simplest model GLM(PCA + G) where the association between a phenotype and markers is directly detected. The pink line represents
the observed p-values using the MLM (PCA + K + G) model. The green line represents the observed p-values using the FarmCPU model. G, genotype
(fixed); PCA, three principal components (fixed); and K, kinship matrix (random).

development was tricky due to bigger blight merging with leaf spots,
so for the analyses, we used the third DS score for both GLS and
NCLB. The association mapping panel was also evaluated in natural
hot spots for GLS and NCLB, and scoring was performed only once
at a grain-filling stage when clearly distinguished GLS and NCLB
symptoms were observed. Therefore, the collected DS data represent
the real response of these lines to the respective diseases.
Nevertheless, both diseases appearing at the same growth stage
and in the same experiment can lead to some confounding effects.
Most of the lines in both the DH population and the association
panel fall into the categories of resistant and moderately resistant,
with a few in moderately susceptible but none in the completely
susceptible category (Figure 1). Overall, the phenotypic data in this
study showed a normal distribution for NCLB and GLS DS scores in
both the biparental CML511×CML546 DH population and the
association panel which supports the quantitative nature of
resistance in these diseases (Nyanapah et al., 2020). The parental
line CML511 exhibited a moderate level of resistance to GLS
congruent with the observations of Kibe et al. (2020a). We also
observed significant genotypic and genotype × environment
interaction variance and moderate-to-high heritabilities in both
the DH population and the association panel, indicating good
prospects for introgressing GLS and NCLB resistance in breeding
programs. Observed heritability estimates for GLS, NCLB, and other
agronomic traits in the DH population are consistent with earlier
studies (Wisser et al., 2011; Benson et al., 2015).
4.1 Trait correlations
There were moderate correlations in GLS and NCLB DS scores
for the biparental DH population between the field trials,
indicating that trait expression was relatively consistent between
the evaluated locations (Supplementary Table S2). On the other
hand, a significantly negative correlation was observed between DS
data of GLS and NCLB, with the flowering time traits AD and SD in
this study (Figure 2). This implies that lower values for AUDPC
(implying higher levels of disease resistance) corresponded with
longer AD or SD. Such negative correlations have also been
reported in other studies (Asea et al., 2009; Wisser et al., 2011;
Kolkman et al., 2020). On the contrary, some studies reported a
positive correlation between GLS resistance and flowering time
(Balint-Kurti et al., 2008; Zwonitzer et al., 2010; Benson et al., 2015;
Mammadov et al., 2015; Liu et al., 2016). This suggests the cautious
use of flowering time in the selection of lines for resistance to GLS
and NCLB.

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TABLE 3 Chromosomal position and SNPs significantly associated with GLS disease severity (GLS_DS) and northern corn leaf blight disease severity (NCLB-DS)
detected by SNP-based GWAS in the association mapping panel.

SNPa Chr MLM MAFb H&Bc Effect Candidate Gene annotation

p-value p-value gene
GLS-DS

S4_170027069 4 8.68E-09 0.35 0.00 0.16 Zm00001eb189650 K13120—protein FAM32A (FAM32A)

S8_155438805 8 1.86E-08 0.47 0.00 −0.20 Zm00001eb360540 Cation efflux protein

S5_214099678 5 7.99E-08 0.29 0.01 0.18 Zm00001eb254100 Zinc finger FYVE domain-containing protein

S10_112359288 10 3.09E-06 0.13 0.13 −0.20 Zm00001eb421180 Copper transport protein atox1-related (abiotic stress)

S2_29666484 2 4.25E-06 0.38 0.13 −0.13 Zm00001eb077270 Wall-associated receptor kinase galacturonan-binding
domain (defense)

S1_253381765 1 4.51E-06 0.40 0.13 −0.13 Zm00001eb049550 Sel-1-like protein

S9_130213878 9 4.53E-06 0.10 0.13 −0.24 Zm00001eb393380 No associated annotations

S2_55483916 2 4.53E-06 0.26 0.13 0.13 ZM00001EB083120 No associated annotations

S1_192041854 1 1.98E-05 0.26 0.51 0.15 Zm00001eb034870 DNA damage–repair/toleration protein (plant defense)

S5_208091867 5 2.37E-05 0.21 0.55 0.12 Zm00001eb251710 Brevis radix domain/regulator of chromosome condensation
(plant defense)

S5_2923669 5 8.36E-05 0.24 1.00 0.13 Zm00001eb211960 NAD-dependent epimerase/dehydratase//cinnamoyl-COA

reductase-like

NCLB-DS

S8_157881780 8 4.41E-15 0.15 0.00 −0.57 GRMZM2G059590 Uncharacterized protein LOC103636219

S8_12990030 8 1.07E-09 0.16 0.00 −0.27 Zm00001d008560 No associated annotations

S4_212315234 4 1.11E-09 0.16 0.00 0.25 Zm00001eb201110 ATP-binding cassette transporter//ABC transporter (plant
defense)

S6_147054037 6 1.75E-07 0.08 0.01 0.25 Zm00001eb285130 TPR repeat-containing THIOREDOXIN TDX

S8_54094984 8 2.11E-07 0.49 0.01 −0.17 Zm00001eb341620 Thioesterase superfamily member-related

S1_194762510 1 6.65E-07 0.22 0.03 −0.26 Zm00001eb035640 AUX/IAA protein//B3 DNA-binding domain//auxin
response factor//DNA-binding pseudobarrel domain (plant
defense)

S1_280826386 1 1.48E-06 0.28 0.05 0.17 Zm00001d034003 Seed maturation family protein

S9_153843575 9 3.08E-06 0.45 0.09 0.16 Zm00001eb400490 Pre-mRNA-processing factor

S5_83980678 5 3.39E-06 0.14 0.09 0.22 Zm00001eb232660 Helicase superfamily/ATP-binding domain (plant defense)

S1_18630129 1 1.05E-05 0.45 0.24 −0.13 Zm00001eb006570 WD and tetratricopeptide repeats protein 1 (WDTC1)

S6_146813774 6 1.64E-05 0.06 0.34 −0.21 Zm00001eb285080 Protein kinase domain (plant defense)

S3_173387708 3 3.49E-05 0.09 0.67 −0.21 Zm00001eb144960 Lipoxygenase (plant defense)

S2_923555 2 4.77E-05 0.32 0.79 −0.16 Zm00001d001787 Cleavage and polyadenylation specificity factor subunit 5
(plant defense)

S7_155701108 7 4.77E-05 0.14 0.79 0.21 Zm00001d021552 Protein of unknown function

S4_233626821 4 5.87E-05 0.47 0.88 −0.15 Zm00001eb204230 Voltage- and ligand-gated potassium channel

S7_8047716 7 6.13E-05 0.24 0.88 −0.12 Zm00001d018877 Plastocyanin-like domain (Cu_bind_like)

a
The exact physical position of the SNP can be inferred from the marker’s name, for example, S5_51353429: chromosome 5; 51353429 bp (Ref Gen_v2 of B73).
b
Minor allele frequency. Candidate gene names are italicized.
c
False discovery rate calculated by using the Benjamini and Hochberg correction method.

In Kenya and Uganda, the main maize-growing area is the major concern which are probably due to the synergistic
frequently affected by GLS and NCLB pathogens (Borchardt interactions of both pathogens. However, a weak correlation was
et al., 1998; Nsibo et al., 2021). When both pathogens affect the observed between GLS DS and NCLB DS. One of the QTLs
maize at the same time, more pronounced necrotic symptoms are identified for GLS (qGLS2_30) was in proximity with the QTL

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Omondi et al.
FIGURE 4
Box-whisker plots for the accuracy of genomic predictions assessed by five-fold cross-validation within association and DH population. AD, days to
anthesis; SD, days to silking; PH, plant height; EH, ear height; GLS, gray leaf spot; AUDPC, area under the disease progress curve; NCLB, northern corn leaf
blight.
identified for NCLB (qNCLB2_220). In the comparison of SNPs
associated with GLS and NCLB DS in association mapping, it was
observed that two SNPs (S1_194762510 and S1280826386)
associated with NCLB DS were collocated within GLS QTL
(qGLS1_54) (Tables 2, 3). This suggests that there are some
common regions contributing to resistance for both diseases. On
the other hand, the observed weak correlation between the DS of the
two diseases could be attributed to the different infection strategies
of the associated pathogens. Cercospora zeina is an apoplastic
necrotroph and a hemibiotroph, while E. turcicum is apoplastic
but then enters the vascular system of the leaf (Kotze et al., 2019).
They also exploit different pathogenicity factors in causing disease
symptoms (Swart et al., 2017; Human et al., 2020).
4.2 QTLs associated with GLS resistance
Most of the QTLs detected for GLS DS were also detected for
GLS AUDPC (Table 2). This was well supported by the observed
strong correlation (r = 0.99) between GLS DS and GLS AUDPC
(Figure 2). A major QTL for GLS resistance (DS and AUDPC),
qGLS1_186, which explained 15.16% of the phenotypic variance,
overlapped with qGLS1_185 which also explained 9.01% of the
phenotypic variance. Intriguingly, this major QTL has favorable
alleles from the donor parent CML511. Kibe et al. (2020a) using the
CML550×CML511 DH population also detected major effect QTL
qGLS1-155 which was located within the physical position of
154–157 Mbp which overlapped with the QTL detected in the
present study (qGLS1_185) spanning between 143 and
185 Mbp. Sun et al. (2021) also fine-mapped a major effect QTL
at the 187–189 Mb region, and the reported flanking markers would
be useful to validate in tropical germplasm.
The chromosome bin 1.06 was described as a QTL hotspot for
GLS resistance as many studies reported earlier (Lehmensiek et al.,
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10.3389/fgene.2023.1282673
2001; Shi et al., 2007; Balint-Kurti et al., 2008; Berger et al., 2014; He
et al., 2018; Lopez-Zuniga et al., 2019; Sun et al., 2021). The
chromosome bin 1.06 also harbors resistance genes to several
other diseases like common rust, southern leaf blight (SLB), ear
rot, and NCLB (Freymark et al., 1993; Wisser et al., 2006; Chung
et al., 2010b; Zwonitzer et al., 2010; Poland et al., 2011; Jamann et al.,
2015). Chung et al. (2010b) demonstrated that the NCLB resistance
QTL at bin 1.06 was important to protect the host against fungal
penetration of E. turcicum using an introgression line population.
The chromosomal region has also been associated with effects on
diverse traits such as grain yield and its components, anthesis silking
interval, and root and shoot traits under both water stress and
optimal water environments (Ribaut et al., 1996; Tuberosa et al.,
2002; Landi et al., 2010). The concentrated mapping of QTL for
several traits, including multiple disease resistance in this
chromosomal region, provides breeders and geneticists an
opportunity to dissect them further and find tightly linked
flanking markers so that this region can be utilized to develop
cultivars with multiple disease resistance.
The qGLS2_30 QTL identified in this study in the chromosomal
bin 2.04 overlapped with QTL reported in earlier studies using
different mapping populations (Balint-Kurti et al., 2008; Lennon
et al., 2017). The QTL qGLS3_151 is placed between 150 and
157 Mbp in the chromosomal bin 3.05, which has previously
been identified as conditioning resistance to SLB and GLS
(Zwonitzer et al., 2010; Kump et al., 2011). The QTL qGLS7_158
positioned between 157 and 159 Mbp in the chromosomal bin
7.04 was also previously reported by Berger et al. (2014) for GLS
resistance. Another QTL qGLS5_16 in the chromosomal bin 5.03 is
also known to have several reported markers for GLS resistance in a
number of association mapping studies (Bubeck et al., 1993;
Clements et al., 2000; Lehmensiek et al., 2001; Shi et al., 2007;
Zhang et al., 2012; Benson et al., 2015). Overall, many of the QTLs
detected in the present study overlapped between the biparental
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Omondi et al.
CML511 × CML546 DH population and the association panel as
well as earlier studies (Supplementary Table S5). The major effect
QTL for both GLS on chromosome 1 is of immediate interest to be
used in resistance breeding.
Among the nine candidate genes identified for GLS resistance
through association mapping, one on chromosome 2
(Zm00001eb077270) encodes a putative receptor-like protein
kinase which are transmembrane signaling proteins that are able
to sense changes in the extracellular environment such as pathogen
invasion (Decreux et al., 2006; Qi et al., 2023). Another candidate
gene on chromosome 1 (Zm00001eb034870) encodes DNA
damage–repair/toleration protein that harbors a leucine-rich
repeat domain which serve as the first line of defense in response
to pathogen-associated molecular patterns (Ng and Xavier, 2011).
The candidate gene Zm00001eb144960 on chromosome 3 encodes
lipoxygenases which are known to be associated with pest resistance,
response to wounding, and plant defense mechanisms where it was
reported to be involved in the early response to pathogen attack
(Peng et al., 1994). Overall, the detected candidate genes in the
association study have annotations inferring direct or indirect
involvement in plant defense.
4.3 QTL and SNPs associated with resistance
to NCLB
This study identified 13 QTLs for NCLB DS and nine QTLs
for NCLB AUDPC. Three of these QTLs were common for both
the DS and AUDPC NCLB traits. The first example of this was
QTLs qNCLB3_50 and qN_AUDPC3_50 that co-localized in the
same position in bin 3.04. This significant QTL for NCLB
resistance has favorable alleles from the parent CML546 (the
more resistant parent). Previous research reports also identified
bin 3.04 as a QTL hotspot conditioning resistance to multiple
diseases including NCLB, SLB, and GLS (Lehmensiek et al., 2001;
Wisser et al., 2006; Shi et al., 2007; Zwonitzer et al., 2010; Kump
et al., 2011; Liu et al., 2016; Lennon et al., 2017; Martins et al.,
2019).
The QTL qNCLB5_83 was positioned in the chromosomal bin
5.04. According to Miedaner et al. (2020), up to eight QTLs have
been localized in this bin showing the importance of this region
for NCLB resistance. Interestingly, the SNP identified through
association mapping (S5_83980678) is positioned within this
QTL region (Table 2, 3). It is associated with a candidate gene
(Zm00001eb232660) that encodes a DNA helicase/ATP-binding
domain. This type of domain has a catalytic function in
unwinding of the double-stranded DNA that is instrumental
in the repair of damaged DNA and DNA replication (Koonin,
1993).
Similarly, qNCLB6_153 for NCLB DS overlapped with qN_
AUDPC6_153 for the AUDPC on chromosome 6 (bin 6.05). Up
to four QTLs were reported from different studies in the same
region (Miedaner et al., 2020). Another pair of QTL, qNCLB8_
171 and qN_AUDPC8_171, corresponded with a previously
reported NCLB resistance QTL by Galiano-Carneiro et al.
(2021). Interestingly, none of the NCLB DS QTLs detected in
this study were found in the same position with chromosomal
Frontiers in Genetics
10.3389/fgene.2023.1282673
bins associated with the qualitative Ht genes (Galiano-Carneiro
and Miedaner, 2017).
An association study on NCLB revealed a significant marker
linked to a candidate gene Zm00001eb201110 on chromosome
4 which encodes for an ATP-binding cassette (ABC) transporter.
Plant proteins with this function are known to be associated with
resistance to fungal and bacterial pathogens through the
transmembrane transport of jasmonic acid or antimicrobial
secondary metabolites (Zhang et al., 2020). Using GWAS,
many studies showed the association of ABC transporter genes
with NCLB resistance (Poland et al., 2011; Ding et al., 2015).
Another candidate gene, Zm00001eb285080, on chromosome
6 encodes a protein kinase, a function known to be important
in regulating the response of plants to pathogen attack (Lehti-
Shiu and Shiu, 2012). There is strong evidence that protein
kinases play a pivotal role in resistance to NCLB (Poland
et al., 2011; Ding et al., 2015; Kolkman et al., 2020).
4.4 QTLs associated with agronomic data
The major QTL for flowering time qAD8_137 was collocated
with QTLs qSD8_137 for SD, qPH8_129 for PH, and qEH8_128 for
EH ((Supplementary Table S4). These QTLs also explained the
major effect of phenotypic variance of 15.8%, 21.4%, 22.39%, and
22.98% for AD, SD, PH, and EH, respectively. Several studies also
recognized chromosomal bin 8.05 as a hotspot for flowering time
QTL and genes (Balint-Kurti et al., 2008; Buckler et al., 2009; Van
Inghelandt et al., 2012). Interestingly, two qualitative resistance
genes, Ht2 and Htn1, were also detected on the same
chromosomal bin 8.05 (Galiano-Carneiro and Miedaner, 2017;
Hurni et al., 2015). The genetic mechanisms underlying flowering
time in this study were largely characterized by additive gene action.
These results agree with the findings of Buckler et al. (2009) who
reported that variations in days to flowering are due to the joint
effect of many minor QTLs with additive effect.
Intriguingly, some of the QTLs associated with flowering time
overlapped with the NCLB and GLS resistance QTL. For instance,
qAD1_60 for flowering time shared the same flanking markers as
qGLS1_54 (Table 2; Supplementary Table S4), and the two SNPs
(S1_192041854, S1_253381765) for GLS DS and two SNPs (S1_
194762510, S1_2800826386) for NCLB DS detected through
association mapping are also positioned in this region.
Another QTL qGLS9_129 also had the same flanking markers
as qAD9_130, and one SNP from association mapping (S9_
130213878) was also detected in the same region for GLS DS.
The NCLB QTL qNCLB1_230 overlapped with qAD1_227 on the
maize chromosomal bin 1.07 (Table 2; Supplementary Table S4).
The QTLs qN_AUDPC2_188 and qPH2_176 overlapped on
chromosomal bin 2.06, sharing the flanking markers. This was
further supported by the positive correlation between PH and
NCLB AUDPC (Figure 2). On the other hand, Galiano-Carneiro
et al. (2021) reported a negative correlation between PH and
NCLB DS. There were no common QTL regions identified for PH
and EH that spanned the same chromosomal regions as GLS DS
and AUDPC. This is supported by the observed negative
correlation between the two traits (Figure 3).
12 frontiersin.org
Omondi et al.
4.5 Genomic prediction of disease and
agronomic traits
Compared to the association panel, the high-prediction
correlations in the DH population for GLS and NCLB could
be attributed to higher similarity or relatedness of individuals
between the training set and the prediction set (Figure 4; Lorenz
and Smith, 2015). GLS DS and GLS AUDPC exhibited slightly
higher prediction accuracies compared to NCLB DS and other
agronomic traits. Agronomic traits, such as AD, SD, PH, and EH,
are characterized by more complex genetic networks, under the
control of numerous QTLs, and affected by the influence of the
environment (Wallace et al., 2014). This presents a challenge in
improving them through phenotypic selection (Du et al., 2021).
Kibe et al. (2020a) reported low-to-moderate prediction
correlations within populations and high values when different
related populations were combined and used in prediction.
Similarly, Galiano-Carneiro et al. (2021) reported prediction
accuracies of moderate levels (0.55) for prediction within
families. Technow et al. (2013) reported prediction accuracies
of 0.58 and 0.55 when using a small population size of
75 individuals. It has been reported that there is no difference
among hybrids advanced through the genomic selection or
phenotypic selection in their response to NCLB and GLS, with
the genomic selection being relatively cheaper than the
phenotypic selection (Beyene et al., 2019; 2021). Overall, the
use of genomic selection has potential to improve the resistance
to GLS and NCLB in breeding populations and could lead to the
development of multiple disease-resistant lines and hybrids.
5 Conclusion
GLS and NCLB are the major biotic stresses that hinder maize
production in high-yielding maize-growing areas in East Africa,
such as western Kenya. The use of genomic tools can provide
useful information to fast track the development of disease-
resistant varieties. In this study, we aimed to identify and
validate genomic regions associated with GLS and NCLB
resistance in biparental and association mapping populations
evaluated in multiple locations in western Kenya. We identified
10 and 11 QTLs for GLS resistance and 18 and 16 QTLs for NCLB
resistance in the DH population and association mapping
population, respectively. We detected a major QTL for GLS
resistance, qGLS1_186, which explained 15.2% phenotypic
variance and qNCLB3_50 for NCLB resistance, explaining
8.8% of the phenotypic variance. Several common QTL
regions between linkage mapping and association mapping
and between NCLB and GLS AUDPC traits were detected. A
negative correlation between flowering time and severity of the
two diseases was reported. Several QTLs identified in the present
study were also co-localized with the QTL previously mapped for
GLS and NCLB resistance. Our study highlights that the
combined use of linkage mapping and genomic selection is an
effective strategy for the improvement of resistance. Genomic
prediction sheds light on new ways to improve breeding for
disease resistance with optimum allocation of resources and lays
the foundation for a new era of resistance breeding.
Frontiers in Genetics
10.3389/fgene.2023.1282673
Data availability statement
The datasets presented in this study can be found in online
repositories: data.cimmyt.org/dataset.xhtml?persistentId=hdl:
11529/10548956 and zenodo.org/records/10046213.
Author contributions
DO: conceptualization, data curation, formal analysis, validation,
and writing–original draft. MD: conceptualization, funding acquisition,
project administration, resources, supervision, and writing–review and
editing. DB: conceptualization, methodology, resources, supervision,
and writing–review and editing. YB: funding acquisition, investigation,
project administration, supervision, and writing–review and editing.
DN: conceptualization, formal analysis, methodology, and
writing–review and editing. CJ: data curation, formal analysis,
methodology, and writing–review and editing. SM: investigation and
writing–review and editing. MG: data curation, formal analysis,
methodology, software, visualization, writing–original draft, and
writing–review and editing.
Funding
The authors declare financial support was received for the research,
authorship, and/or publication of this article. This study was supported
by the Kenya–South Africa joint science and technology research
collaboration. The grants received from the National Research Fund,
Kenya, and the National Research Foundation (NRF), South Africa
(grant # 105806), toward this research are hereby acknowledged. This
study was also supported by CIMMYT-Nairobi. CIMMYT received
support from the United States Agency for International Development,
Foundation for Food and Agriculture Research (FFAR), and the Bill and
Melinda Gates Foundation (BMGF) under AG2MW (Accelerating
Genetic Gains in Maize and Wheat for Improved Livelihoods,
B&MGF Investment ID INV-003439) project, the CGIAR Research
Program on MAIZE.
Acknowledgments
The authors thank the CIMMYT field and laboratory technicians
for phenotypic evaluations and sample preparation for genotyping.
They are also grateful to the Buckler Lab at Cornell University,
United States, for genotyping the maize populations and providing
the marker information and Diversity Arrays Technology (DArT),
Canberra, Australia for genotyping DH population. Part of the data in
this manuscript was from the thesis work conducted by DO.
Conflict of interest
Authors DO and CJ were employed by Crop Science Division
Bayer East Africa Limited.
The remaining authors declare that the research was conducted
in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
13 frontiersin.org
Omondi et al. 10.3389/fgene.2023.1282673

The authors declare that they were editorial board members of reviewers. Any product that may be evaluated in this article, or
Frontiers, at the time of submission. This had no impact on the peer claim that may be made by its manufacturer, is not guaranteed or
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Publisher’s note Supplementary material

All claims expressed in this article are solely those of the authors The Supplementary Material for this article can be found online
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